1. Field of the Invention
The invention relates to methods for separating components from blood or plasma and, more specifically, the invention relates to methods with improved recovery of the blood or plasma component.
2. Description of the Related Prior Art
WO 96/16714 discloses a container for separating a blood or plasma components, e.g., fibrin monomer, from blood or plasma by a centrifugation about a vertical axis. This container comprises a first annular chamber defined by an outer cylindrical wall and an inner cylindrical wall, both walls extending coaxially about a common axis, as well as by a top wall and a bottom wall, where the bottom wall is formed by a piston displaceable within the first chamber. The container further comprises a second chamber accommodated below the first chamber and communicating with the first chamber through a first conduit. The second chamber is defined by the outer cylindrical wall, the bottom wall of the first chamber, and by a second bottom wall. This second chamber serves as reaction chamber for receiving plasma and treating the plasma to obtain the desired component. For example, treatment of plasma fibrinogen with thrombin or a thrombin-like enzyme converts the fibrinogen to fibrin monomer which spontaneously polymerizes to a non-crosslinked fibrin polymer.
Placing this container in a centrifuge for the above-described reaction provides that the non-crosslinked fibrin polymer is separated from the plasma and deposited on an outer wall of the reaction chamber during centrifugation. When the piston is subsequently actuated, the remaining plasma is removed from the reaction chamber. Thereafter, a solvent is added for dissolving the so-deposited non-crosslinked fibrin polymer and forming the desired fibrin monomer solution.
As described in detail in EP 592242, this fibrin monomer solution is extremely useful, for example, in fibrin sealant methods. It is desirable to use devices like those described in U.S. Pat. No. 5,603,845, WO 96/16713, U.S. Pat. No. 5,935,432, WO 96/16714, U.S. Pat. No. 5,824,230, WO 96/16715 and U.S. Pat. No. 5,733,446, the disclosures of which are all incorporated herein by reference, to prepare blood products such as fibrin sealant components immediately at the time of surgery so that autologous blood can be utilized. It may also be desirable from a surgeon""s perspective to use sealant products which are relatively uniform from one procedure to another. This is nearly impossible for freshly prepared products, however, since fibrinogen concentration in human blood can vary by xc2x1300% in human patient populations and freshly prepared sealant components from individual sources will also vary. Most humans have fibrinogen levels between 2 and 6 mg/ml of plasma and some humans may have as little as 1 mg/ml and some as much as 10 mg/ml (fibrinogen plasma).
As described in WO 98/30887 and U.S. Pat. No. 5,955,026, the disclosures of which are incorporated herein by reference, a method and apparatus are known which involve introducing blood or plasma into a container having a light transmissive wall and providing a reaction which results in a polymerized form of the component being deposited into the wall. An optical reading of the difference in light transmission through the wall alone and the wall with polymer thereon can be related to the total amount of the component from the blood or plasma sample.
Used in this manner the apparatus and methods disclosed in WO/30887 and U.S. Pat. No. 5,955,026 are useful to determine the concentration of the component in the blood or plasma. Further, by knowing the amount of solvent or buffer to be used to solubilize the polymerized component to yield the desired component solution, the concentration of the resulting solution is readily available. Still further, when the optical determination of the concentration of the component in blood or plasma (or the determination of the amount of polymerized component) is made, this data can be used to control the amount of buffer or solvent used to solubilize the polymer to prepare solutions of desired concentrations. Still further, when the buffer or solvent is also used so that the resulting solutions are of a specific value or range of values of pH, limits on the minimum and maximum amount of buffer or solvent used can be employed. For example, when a plasma fraction is reacted to form a fibrin polymer and a pH-4 acetate buffer is used to solubilize the polymer to form a desired fibrin monomer solution, minimum and maximum amounts of buffer can be programmed into the process and apparatus to keep the resulting pH within a desired range, e.g., 4.0-4.5. The methods described in WO 98/30887 can provide fibrin monomer solutions of about 20 mg/ml xc2x125% while simultaneously maintaining pH value between 4.0-4.5. This represents a remarkable 10-fold increase in reproducibility of freshly prepared or autologous fibrin monomer solution. It is also important to note that the optical sensing apparatus and methods described in WO 98/30887 determine amounts/concentrations of components in a container rotating at high speeds, e.g., up to 9000-10,000 RPM rather than taking an optical measurement in a fixed position.
Apparatuses and methods such as those discussed above, while advantageous, may still not recover all of the fibrin polymer deposited on the container walls. Accordingly, it would be advantageous to ensure removal of all or substantially all of the polymerized form of the component deposited on the wall of the container in order to maximize the amount of recovery of the component.
Accordingly, an object of the invention is to provide a method whereby supply of solvent is controlled and, in particular, solvent is applied in multiple steps to maximize recovery of the polymerized form of the desired component.
In order to achieve the foregoing object and other objects, there is provided a method for preparing a solution of a blood or plasma component with improved recovery of the blood or plasma component. The method in accordance with the present invention includes the step of subjecting blood or plasma to conditions for catalyzing formation of a polymerized form of said component of said blood or plasma. The polymerized form of the component is then recovered, and an initial amount of a solvent solution is added to the recovered polymerized form of the component to dissolve it. The initial amount of solvent is an amount less than a predetermined total amount of buffer solution for dissolving all of the recovered polymerized form of the component. Additional solvent is added to the recovered polymerized form of the component. The additional solvent is added to the recovered polymerized form of the component in an amount equivalent to a difference between the predetermined total amount of solvent and the initial amount of solvent added to dissolve the polymerized form of the component.